Yan Jiang ,Hui Qi,Xianming Zhang ,*

1 Engineering Research Centre for Waste Oil Recovery Technology and Equipment,Ministry Education,Chongqing Technology and Business University,Chongqing 400067,China

2 College of Foreign Languages,Chongqing Technology and Business University,Chongqing 400067,China

1.Introduction

The lubricant consumption in China is the 2nd in the world.Lubricant hastens the rapid development of modern industries at the cost of prevalent environmental oil pollutions due to accidental leaking and improper disposal[1,2].It is reported that discharges of used lubricating oil in land and waterway account for about 40%of total oil inputs,and considering the restrictions of recycling channels and regeneration techniques,the discharge is far more than that.The remediation of oil-contaminated sites raises a growing concern in many countries[3,4].

Physical and chemical methods are widely employed for lubricant treatment,but these methods are often grossly inadequate and ineffective,easily causing secondary pollution[5].Biological techniques have been developed as an efficient,economic and eco-friendly treatment[3,6].Leeet al.[7,8]focused on biodegradation of specific substrates of two recalcitrant lubricantingredients,cyclohexane and hexane,with the maximum specific degradation rate of246 and 361μmol·(g DCW·h)-1,respectively,byRhodococcussp.isolated from oil-contaminated soil.The strain could utilize many other hydrocarbons under optimal conditions,such as PAHs and BTEX.Khondeeet al.[2]made use of a bioreactor containing chitosan-immobilizedSphingobiumsp.P2 to remove automotive lubricants from emulsified wastewater.It is worth noting that an internal loop airlift bioreactor and immobilized cells were utilized in the tests,which are regarded as the advanced technologies in the field.However,the removal rate of 200 mg·L-1total petroleum hydrocarbons only reaches 80%-90%.These studies mainly concentrate upon two aspects,specific substrate biodegradation and bioremediation of contaminated sites and wastes by petroleum hydroxycarbons,with the microbial strains.Currently,many microorganisms with the ability to degrade crude oil,diesel oil and heavy oil,as well as their compositions have been isolated and identified from pollution sites and oily sludge.They are usually screened from a large quantity of isolates by traditional shaking flask tests with such disadvantages as chemical consumption,heavy load and long cycle.Since biodegradation occurs in oil-water phase,well plate method would be preferred for high-throughput isolate screening.Lubricating oils float on water,so it is easy to evaluate the biodegradation through oil film change.Presently,well plate tests have been applied in fermentation experiments for different purposes,but seldom employed in oil-water systems[9].

It is a growing trend to add exogenous microorganisms in bioremediation,which hinges on bacterial activity for lubricating oil degradation.As the ingredients in lubricating oil are a complex mixture of hydrocarbons including linear and branched paraffins,cyclic alkanes and aromatic hydrocarbons,activity of effective lubricant-degrading microorganisms reflects their biodegradation abilities for multisubstrate in the aspect of lubricant biodegradation[10].Study on lubricating oil as substrate is of practical significance for the treatment of lubricating oil contamination.

The objectives of this study are to isolate and identify lubricantdegrading bacteria from used lubricating oil and acclimated oilcontaminated soil separately,to investigate different methods on bacterial isolation and explore the possibility of well plate method in high-throughput screening,to describe bacterial biodegradation characterization by shaking flask experiments,and to evaluate lipase activity by comparison of two different methods.

2.Materials and Method

2.1.Acclimation of soil sample

Soils perennially contaminated by lubricating oil were collected from a used lubricant recycling enterprise in Chongqing,China.Soils were sampled 0-20 cm underneath the regolith,dried at 37°C in a constant temperature incubator to be easily comminuted,and sieved to 2 mm.Then it was enriched acclimation at 37°C using lubricating oil as sole carbon source for a period of 37 days.In order to avoid the death of indigenous microorganisms owing to the side effect of highconcentration petroleum hydrocarbons,30 g soil was reserved as isolation sample according to five-point sampling method before each gradient acclimation.Table 1 summarizes the acclimation method.2.5%and 5.0%urea and 0.2 mol·L-1dipotassium phosphate were served as fertilizer to provide nutrients N and P in order to stimulate indigenous microorganisms[11].Their addition depended on lubricant concentration in soil and acclimation stage[12].Besides,moisturizing and turning over the soil were conducted twice every day.In samples 3 and 4 with high-concentration lubricating oil,0.5%(v/v)Tween 80 was introduced as surfactant in order to enhance indigenous microorganism biodegradation.

2.2.Bacterial isolation,identification and cultivation

Bacterial strains were isolated from acclimated soil and used lubricating oil.5 g soil and lubricating oil were inoculated into shaking flasks with LB medium(peptone 10 g·L-1,yeast extract 5 g·L-1,sodium chloride 10 g·L-1)separately.After three enrichment cultures,the cultures were inoculated into mineral salt medium(MSM),composed of(g·L-1):1 NH4NO3,0.8 K2H0PO4,0.4 KH2PO4,0.8 NaCl,0.05 CaCl2,0.05 MgSO4and 0.05 FeSO4,and 150 mg·L-1lubricating oil was used in this test.The initial pH was adjusted to 7.2 before sterilization at121°C for 20 min.Incubation was done in a rotary shaker with 200 r·min-1at 37 °C.After two selective cultures,100 μl cultures were plated onto agar plates.In order to improve the likelihood to obtain highly active strains and decrease the disturbance from untargeted strains,such methods were designed aiming at the plates as LB,LB and MSM containing lubricant,and LB and MSM with oil film on the surface for the isolation from soil sample,and LB,LB and MSM with oil film on the surface for the isolation from lubricant sample.In each of the eight methods,5 plates were continually coated.The individual colonies were inoculated on nutrient agar slants and evaluated for the potential to degrade lubricating oilthrough 24-well plates and shaking flask tests.

The isolates screened were identified based on 16S rRNA sequencing(Shanghai Sunny Biotechnology Co.,Ltd,China)followed by BLAST analysis(NCBI)with closed match with available sequences.

2.3.24-Well plate tests

The isolates from slants were cultivated overnight in 10 ml LB medium.0.4 ml of this culture was inoculated to 20 ml fresh medium.Subculture with OD600=1.2±0.02 was harvested to inoculate into oil-water system.Immediately after measurements of optical density,this subculture was injected into 1.5 ml sterilized centrifugal tubes to centrifuge for10 min at6000 r·min-1and the supernate was discarded.Cells were re-suspended using 1.5 ml phosphate buffer(pH=7.2).After cells were washed,cell suspension was centrifuged.The operation above was repeated twice.Resting cells were prepared by cell resuspension with 0.2 ml phosphate buffer,which also served as inoculum to conduct well plate tests.

2.5 ml MSM was added into sterile 24-well plates,0.2 ml different inoculum was inoculated in each hole,and immediately 0.3 ml used lubricating oil was combined.24-well plates were cultivated for 5 days for three subcultures as inoculum and 7 days for three resting cells at 37°C.Samples without inoculation and without oil were used as two different controls.All tests including controls were repeated three times.Biodegradation of isolates was evaluated mainly based on the change of oil film supplemented with cell density determination.

2.4.Shaking flask tests

A total of 15 isolates were obtained after preliminary screening and further tested by shaking flasks.2%(v/v)subculture(OD600=1.2±0.02)as inoculum was inoculated into shaking flasks supplemented with 25 ml MSM and lubricant concentration of 100 mg·L-1for 30 h cultivation at37 °C and 200 r·min-1in a rotary shaker.In order to compare and choose the more effectiveAcinetobacter johnsonii,a series of shaking flasks were applied to evaluate their biodegradation for 400 and 800 mg·L-1lubricating oil at 5%inoculum volume.Moreover,5 screened isolates were tested for the biodegradation potential at1000 mg·L-1lubricant and limiting concentration.Residual lubricant concentration and cell density were determined.

Table 1Acclimation of soil sample

2.5.Enzyme assay

Bacterial lipase activity was determined by two methods,indicator titration method and ultraviolet spectrophotometry.

2.5.1.Indicator titration method

This method is PRC National Standard of Lipase Preparations(GB/T 23535-2009),described as follows.4%polyvinyl alcohol(PVA)was filtered using double-layer gauze,and filtrate was collected and combined with 1/3 volumetric olive oil.The mixture was stirred at 40°C for approximately 5 min using a magnetic stirrer to prepare PVA emulsion.

In enzyme assay,4.00 ml PVA emulsion and 5.00 ml phosphate buffer(pH=7.5)were added in turn to two 100 ml conical flasks numbered control A and sample B,then 15 ml 95%ethyl alcohol was added into control A.Preheated in water bath at 40°C for 5 min,the flasks were inoculated with 1 ml cell culture supernatant separately,immediately shaken up and reacted for 15 min.The reaction in sample B was terminated by adding 15.0 ml 95%ethyl alcohol.Reaction liquids were titrated using standard sodium hydroxide solution with phenolphthalein as indicator.Enzyme activity is calculated by

whereX1is the enzyme activity(U·g-1),V1andV2are the volume of sodium hydroxide solution for sample and control(ml),respectively,cis sodium hydroxide solution concentration(mol·L-1),50 means that 1.00 ml 0.05 mol·L-1sodium hydroxide solution is equal to 50 μmol aliphatic acid,n1is the dilution ratio of sample(it is 1 in this test),0.05 is the conversion coefficient of sodium hydroxide solution concentration,and 1/15 is the reaction time with 1 min as standard.

2.5.2.Ultraviolet spectrophotometry

Lipase activity was also determined by a popular method[13,14].Enzyme activity was assessed by hydrolysis ofp-NPP.One unit of lipase(U · ml-1)is defined as the amount of enzyme liberating 1.0 μmolpnitrophenol by hydrolysis ofp-NPP per minute under standard assay conditions with pH 8.0,30°C and 400 nm.

2.6.Analytical method

Cell density was determined spectrophotometrically by measuring the optical density at600 nm[15].Residual lubricating oil concentration in solution was monitored by the modified Standard Examination Methods for Drinking Water—Aggregate Organic Parameters(GB/T 5750.7-2006).20 ml cell culture was transferred into 60 ml pearshaped funnel.After the treatment with 2 ml sulfuric acid solution(volume ratio of 1:1)and 0.5 g sodium chloride,the mixture was extracted twice using petroleum ether(boiling range 60-90°C).Extract liquors were combined into 25 ml volumetric flask and dehydrated using anhydrous sodium sulfate,and then lubricating oil concentration was monitored spectrophotometrically by measuring the absorbance at wavelength 256 nm.OD256value was converted to oil concentration(mg·L-1)according to previous standard regression curve(A=0.00143C-0.0015,r=0.9991).

As the soil contained some vegetable oil compositions,the oil content was monitored by modified gravimetric method in order to eliminate the disturbance from vegetable oil.The modification for vegetable oil removal from dry soil sample by saponification method is summarized as follows.(1)Chloroform extraction.25 g soil sample was extracted by 50 ml chloroform for 16 h.Heated in water bath at 55°C for 1 h,the extract was filtered and the filtrate was collected into 100 ml breaker weighed previously.Soil was extracted twice again using 25 ml chloroform at 55°C for 0.5 h.Combined extract was evaporated to dryness in fume cupboard at 55°C.Then the breaker was dried in drying oven at 70°C for 4 h.The weight increment was the quantity of chloroform extract.(2)Vegetable oil removal with saponification.50 ml potassium hydroxide-ethanol solution at the concentration of 0.5 mol·L-1was added in chloroform extract placed at 70°C water bath to saponify for 1 h under stirring.Saponification solution was transferred into 250 ml separating funnel and the breaker was washed by 50 ml deionized water and 50 ml petroleum ether separately.After the extract of petroleum ether was dehydrated by anhydrous sodium sulfate,the solution was filtered into another previously weighed breaker.(3)Detection of oil content with gravimetric method.It is similar to the first step except for the water bath temperature of 40°C.

All experiments except for Table 1 were repeated three times.The data shown in the corresponding figures and tables are mean values and the error bars present the standard deviation.

3.Results and Discussion

3.1.Acclimation of soil and isolation of microbial species

Soil sample was acclimated by simulating bioremediation and C/N/P ratios reported in literature varied widely[11]due to the effects of different factors,among which the species of indigenous microorganism were regarded as the key factor based on different demands for N and P.In the present study on microbial isolation,the characters of indigenous microorganism were unknown,so N and P were not accurately added and the concentration was controlled within a certain range according to lubricant concentration in soil.Tween 80 was added to accelerate oil mixing,conducive to the contact of indigenous microorganisms with oil.Besides,5.0%urea was used to substitute for 2.5% of that matter because of high use level leading to run-off.Through such acclimation,indigenous microorganisms could promote their lubricant tolerance within a short time and part of untargeted strains might be obsoleted during isolation.

It has been reported that sample acclimations,including wastewater,contaminated soil and activated sludge,can effectively enhance activities of indigenous microorganisms using special substrate as sole carbon[16-18].However,high-concentration lubricating oil may bring about strong toxicity to inhibit cell growth,so such sample can be used as separation source as long as biodegradation functions.As shown in Table 1,biodegradation slowly proceeds in the late stages of gradient acclimation with a little decrease of oil content from 20.51%to 18.85%in stage III and from 29.96%to 29.22%in stage IV.The results indicate lubricant tolerance of indigenous microorganisms,which may be efficient degraders.The lubricant removal was successful in samples 1 and 2,but only samples 3 and 4 were chosen as separation sources after being mixed in order to obtain highly efficient bacteria and lighten workload of screening isolates.

During isolation,two kinds of liquid and solid medium were used,and MSM plates were also applied to bacterial isolation besides LB plates.Furthermore,they were mixed or coated on the surface with lubricant to create extreme growth environment,in which untargeted strains could be efficiently erased.Similar research was also conducted by Kaczoreket al.[19],who studied the difference of cell growth on standard nutrient agar plates and on agar plates with 50 μl diesel oil as the sole carbon and energy source.Their results demonstrated that the diesel oil changed biodegradation potential and biochemical properties,as well as enzyme activity.In this study,those isolates grown on MSM plate with oil film were preferentially selected and a total of 51 individual isolates were obtained.

3.2.Preliminary screening of isolates by 24-well plate test

In this study,gradual vanishing of oil film was used as the criterion to evaluate bacteria biodegradation.Oil film gradually decreased from center to border with the consumption of lubricating oil.The surface color of cell culture turned to oyster white,and finally very little residual lubricating oil distributed over hole border with cell culture turning to turbid in good biodegradation tests.However,a disadvantage of well plate test was found in tests:the results were different for the same isolate,so only the isolates with at least two good results in resting cell tests and one good result in cell culture tests as inoculum were reserved.Those isolates with two good results in cell culture tests and only one good result in resting cell tests were discarded for their weak biodegradation potentials(Table 2),because resting cells presented poor activity by comparison with cell culture.Resting cells reflect bacteria essential attributes more clearly.

Cell density can be regarded as a supplemented criterion to assist isolate screening.2.1 ml cell culture was sampled to determine optical density using 1 cm cuvette.Table 2 shows a regularity that OD600values are consistent with the change of oil film,that is,the higher the cell concentration,the more obviously the oil film disappears.In good biodegradation tests,optical density values were 0.20-0.30 in subculture tests and 0.10-0.22 in resting cell tests with more substrates consumed to overcome substrate inhibition on cells.The phenomenon reflected on not only long biodegradation time but also less biomass.In view of the defect of well plate method,15 isolates were reserved after two repeated tests,which decreased workload.Such sample capacity would not omit target strains but decrease workload.The shortage of this method could be covered by following shaking flask tests.

Table 2Change of oil film and cell density in well plate tests

3.3.Re-screening of isolates by shaking flask tests

15 isolates were evaluated for their potentials to degrade lubricating oil by inoculating each strain into MSM containing 100 mg·L-1lubricating oil as the sole carbon source.In the test,all isolates could not completely utilize the oil in 30 h,which was more accurate to compare biodegradation potential of the isolates.Fig.1 shows three isolates with the lubricant removal rate of about 90%.Considering lubricant component diversity,different microorganisms might have extraordinary potential for special petroleum hydrocarbon,so 6 isolates with the removal rate over 80%were reserved for further study.They would be as materialbasis to construct highly-efficient colonies.The regularity was noticed that lubricant removal rates were consistent with corresponding cell densities for all isolates.Cell cultures of 6 isolates possessed high optical density values with 0.16-0.20.

Fig.1.Biodegradation of 15 isolates for 100 mg·L-1 lubricating oil.

3.4.Identification and biodegradation of microbial species

Gram stain and microscopic examination were conducted on 6 isolates.The results showed that strain 1 from contaminated soil and strain 13 from used lubricating oil presented similar microscopic morphology.Also,lubricant removal rate and cell density were similar in Fig.1.They were regarded as the same and strain 13 was chosen for identification.Similar phenomenon also occurred to strains 6 and 11 isolated from soil and lubricating oil,respectively,while strain 6 retained greater ability to stably degrade lubricating oil(Fig.1).In order to compare their biodegradation,further tests were conducted.

As shown in Fig.2,strain 6 demonstrated higher potential to degrade lubricating oil and took less time to completely degrade 400 and 800 mg·L-1substrates in 83.5 and 125 h,respectively.Moreover,cell concentrations were a bit higher.These manifested that strain 6 had stronger capacity to get rid of lag phase of cell growth than strain 11,because less lubricating oil was utilized to overcome substrate inhibition from toxicity of organic pollutant and more substrates were consumed to synthesize new cells.Another phenomenon was observed that cell concentration only increased from about 0.49 to 0.63 with the two-fold increase of lubricant concentration.In the initial phase of 800 mg·L-1biodegradation system,stronger inhibition was produced with more substrate consumption,while cells grown in the late phase presented large specific degradation rate because of high cell concentration and activity,leading to more rapid biodegradation at the same substrate concentration.It was verified by sharp drop of curve.These results prove that the strains possess different potentials for lubricant degradation although they have similar microscopy examination.Thus strains 6 and 11 are considered as either the same strains with different biodegradation characterization or different strains with similar microscopic morphology.Anyhow,they possess certain value for further study.

Fig.2.Cell growth and lubricant degradation by two A.johnsonii separately isolated from soil and lubricating oil in MSM containing initial lubricant concentration of 400 and 800 mg·L-1 with 5%starting inoculum.

Finally,5 isolates were identified by sequencing the 16S rRNA gene using PCR and universal primers 27 F(5′-AGA GTT TGA TCC TGG CTC AG-3′)and 1492R(5′-CTACGG CTACCT TGT TAC GA-3′).The identification revealed the presence ofAcidovorax citrulli(strain 3),Pseudomonas balearica(strain 4),A.johnsonii(strains 6 and 11)andAcidovoraxavenae(strain 13).Among these five strains,strains 3,4 and 6 were isolated from contaminated soil using solid MSM plated oil film on surface;strains 11 and 13 were from lubricating oil and isolated by solid MSM containing lubricating oil with or without oil film.Strain 1 is regarded as the same with strain 13,so it is not identified.Both strains 6 and 11 areA.johnsonii,and the phylogenetic analysis of their sequences is shown in Fig.3.The two strains show high sequence similarity(99%)to bacteriaAcinetobactersp.WAB 1867,which illustrates that the same strain growing in different environment may have similar or different biodegradation ability for the same substrate(Fig.2).Obviously,microorganisms from acclimated samples and used lubricating oil possess potential to degrade lubricating oil.Those with high activities can rely on growth and non-growth substrates to implement metabolism.Sample acclimation as well as various plate disposals may be effective for isolation of various functional strains.

3.5.Lubricant biodegradation

5 bacteria biodegradations were tested at high lubricant concentration.As shown in Fig.4,1000 mg·L-1lubricating oil could be entirely utilized at37 °C,5% inoculum volume and 180 r·min-1.Among them,P.balearicaandA.johnsoniifrom oil-contaminated soil(strain 6)possessed the highest potential.They could degrade 1000 mg·L-1lubricating oil in about 158 h and the final cell concentrations were similar.Strain 6 took 8 h less to degrade 1000 mg·L-1lubricating oil than strain 11,which again proves that the same strain from different environments may possess different degradation capacities.Two strains ofAcidovoraxsp.are con firmed for their lubricant biodegradation capacities,which would be conducive to future study on acid sludge and soil bioremediations.Biomass with OD value of 0.669-0.701 presents consistent regularity with substrate consumption for these five bacteria,and the faster the biodegradation,the higher the final cell concentration.

Fig.5 describes the five bacterial maximal biodegradation with the initial lubricant concentration of 1500,1850,1900,2100 and 2200 mg·L-1.Their different degradation capacities are clearly reflected in the limiting concentration.The higher the substrate concentration,the longer time the biodegradation lasts.For example,even forA.avenaeandA.johnsoniistrain 11 with the maximum concentration of 1900 and 1850 mg·L-1,respectively,the former spent 3 h more than the latter to degrade entire substrates.For these five strains,biodegradation did not occur when substrate concentration increased by 50 mg·L-1gradient.Compared with Figs.1,2 and 4,the regularity is that at the same substrate concentration the strain with the fastest degradation rate presents the greatest degradation ability.A.johnsoniistrain 6 andA.johnsoniistrain 11 are re-confirmed to possess different degradation potentials,with the maximum degradation capacities of 1850 and 2100 mg·L-1,corresponding to the degradation time of 263 and 272 h,respectively.Still,cell growth is consistent with the substrate consumption,which manifests that the higher the substrate concentration,the higher the final cell concentration,with the drop of cell concentration increase amplitude mainly due to more lubricant consumption to overcome the substrate inhibition.

3.6.Enzyme assay

Many petroleum hydrocarbon degraders rely heavily on excreted lipase to biodegrade lubricating oil,so lipase activity was investigated using two methods.In the indicator titration method,15 isolates were determined for enzyme activity on the basis of NaOH consumption.In Table 3,these isolates show similar lipase activity(part of data not shown),and more importantly,NaOH consumption is very similar.The phenomenon occurs with slight chance because these 15 strains are randomly isolated from two samples.The results are unconvincing,which is attributed to improper detection method.

Fig.3.Phylogenetic analysis of 16S rRNA sequences of strains 6 and 11.

Fig.4.Cell growth and lubricant degradation by 5 strains in MSM containing initial lubricant concentration of 1000 mg·L-1 with 5%starting inoculum.

Thus,ultraviolet spectrophotometry was established to determine these 6 bacterial lipase activities in cell culture,which relies on the hydrolysis ofp-NPP.In tests,because OD400values at low concentration would fluctuate,experimental data were conducted outlier tests using Grubbs'method(α=0.05).Finally,p-NPP standard equation was established by linear regression asA=7.693C+0.00373(F=2478.84).Lipase activities were summarized in Table 3.The strains retained high lipase activity except for two strainsA.johnsonii.StrainP.balearicais proved to have the highest lubricant-degrading potential(Figs.1,4 and 5),but the enzyme activity is 6.03 U·ml-1,which is lower than that of stain 1 andA.citrulli.TwoA.johnsoniistains also present high biodegradation potential but very low lipase activity.It might be interpreted as that manyAcinetobactersp.largely secret lipase under alkaline condition.WhenA.johnsoniiis metabolized,organic acids are produced,decreasing the pH value of cell culture to inhibit lipase activity[20,21].The conclusion is that microorganisms degrading lubricating oil do not absolutely depend on lipase excretion and other degradation mechanism may exist,for example,some microbes could utilize its intermediate metabolites as growth substrates to promote specific biodegradation[22].

Fig.5.Cell growth and substrate degradation by 5 strains in limiting concentration.

4.Conclusions

Sample acclimation and rigorous growth conditions are feasible for isolation of highly active strains,which promotes bacterial tolerance for specific pollutant.In spite of the existence of accidental factors,24-well plate tests can be easily applied in bacterial screening by characterizations of oil film and cell density.The method shortens experimental period and greatly reduces workload,which is especially appropriate for high-throughput screening.The identification of twoA.johnsoniidemonstrates that the same strain derived from different contaminated sites may have different biodegradation potentials for the same substrate.The indicator titration method described in GB/T 23535-2009 is incompetent in the present study,whilep-NPP method can successfully determine lipase activity in cell culture.The results indicate that the two strains ofA.johnsoniiwith high biodegradation potential only put up low enzyme activity.Consequently,lipase enzyme activity cannot be used as criterion to evaluate bacterial biodegradation potentials for petroleum hydrocarbons.

Table 3Lipase activity determined by indicator titration and p-NPP methods

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